Methods for controlling the growth of prokaryotic and eukaryotic cells

ABSTRACT

The present disclosure relates to methods for control of cell growth rates where cell growth is measured in situ. The methods are applicable to bacterial cells, mammalian cells, non-mammalian eukaryotic cells, plant cells, yeast cells, fungi, and archea.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 16/552,981,filed 27 Aug. 2019; Ser. No. 16/360,404, filed Mar. 21, 2019, now U.S.Pat. No. 10,435,662; and Ser. No. 16/360,423, filed Mar. 21, 2019, nowU.S. Pat. No. 10,443,031, both of which claim priority to U.S.Provisional Patent Application No. 62/649,731, filed Mar. 29, 2018, andU.S. Provisional Patent Application No. 62/671,385, filed May 14, 2018;all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure relates to methods for control of cell growthrates and subsequent cell processing. The methods are applicable tobacterial cells, mammalian cells, non-mammalian eukaryotic cells, plantcells, yeast cells, fungi, and archea.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Optical density (OD), measured in a spectrophotometer, can be used as ameasure of the concentration of cells in suspension. As visible lightpasses through a cell suspension, the light is scattered. Greaterscatter indicates that more cells are present. Typically when workingwith a particular type of cell, one determines optical density at aparticular wavelength that correlates with the growth media used. Forbacteria, generally cells are grown in, e.g., LB broth, and OD₆₀₀ ismeasured.

To determine the OD of a cell culture, typically a quartz cuvette isused with a benchtop spectrophotometer. A wavelength is selected on thespectrophotometer, and a cuvette containing a control liquid (e.g., ablank)—almost always the growth medium in which the cells are beingincubated—is inserted into the sample compartment within thespectrophotometer. A transmittance/absorbance control is then set to100% transmittance. Once the control is adjusted for 100% transmittance,turbidity measurements can be made. The blank is removed, and then analiquot of the sample to be measured is pipetted into another cuvetteand the cuvette is inserted into the sample compartment. Thespectrophotometer will then indicate the OD and percent transmittance ofthe sample. One drawback to using this traditional method for measuringOD is that it requires human intervention; that is, aliquots of thesample to be measured must be taken at intervals, loaded into cuvettes,and inserted into the spectrophotometer to get a reading. Not only doesthis procedure require time and effort, but invasively accessing thegrowing cell culture runs a risk that the cell culture may becontaminated. Further, the cell culture is depleted with each sampleremoved. An additional drawback is that once the cells are growing, itis difficult to predict when the cells will reach a target OD.

Accordingly, there is a need in the art for a cell growth device thatnoninvasively, rapidly, predictably and reproducibly promotes growth ina variety of cell types while automatically measuring the OD of thecells in the vessel in which they are growing. Additionally, there is aneed in the art for a cell growth device that controls the growth of thecells to a target OD at a target time as requested by a user. Such acell growth device can function either as a stand-alone device, e.g.,benchtop device, or the cell growth device can be employed as one modulein a multi-module automated cell processing system. The disclosed cellgrowth devices and methods address these needs.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

Provided herein are methods, devices, and instruments for automatedcontrol of cell growth rates where growth of the cells is measured insitu. The devices can be used as a stand-alone device or as one modulein an automated environment, e.g., as one module in multi-module cellprocessing environment. The cell growth device includes atemperature-controlled rotating growth vial, a motor assembly to spinthe vial, a spectrophotometer for measuring, e.g., OD in the vial, and aprocessor to accept input from a user and control the growth rate of thecells. The cell growth devices and associated methods noninvasively,rapidly, predictably and reproducibly promote growth in a variety ofcell types. The methods and devices described herein automaticallymeasure the OD of the growing cells in the rotating growth vialcontinuously or at set intervals and control the growth of the cells toa target OD and a target time as specified by the user. That is, themethods and devices described herein provide a feedback loop thatmonitors cell growth in real time and adjusts the temperature of therotating growth vial in real time to reach the target OD at a targettime specified by a user.

Thus, some embodiments provide a rotating growth vial comprising: avial; a motor assembly configured to connect to a motor and spin thevial; an electrical connection configured to be electrically coupled toa thermal control device; a light path through the vial configured toallow light generated from a spectrophotometer to measure and deliver toa processor a value of a characteristic of cells in the vial; and aconnection with the processor, wherein the processor accepts input froma user, receives from the spectrophotometer the measure of the value ofthe characteristic of the cells, and directs the thermal control deviceto adjust the temperature of the vial to grow the cells in the vial to atarget value at a target time. In some aspects, the rotating growth vialhas two or more “paddles” or interior features disposed within therotating growth vial. In some aspects, the width of the paddles orinterior features varies with the size or volume of the rotating growthvial, and may range from 1/20 to just over ⅓ the diameter of therotating growth vial, or from 1/15 to ¼ the diameter of the rotatinggrowth vial, or from 1/10 to ⅕ the diameter of the rotating growth vial.In some aspects, the length of the paddles varies with the size orvolume of the rotating growth vial, and may range from ⅘ to ¼ the lengthof the main body of the rotating growth vial, or from ¾ to ⅓ the lengthof the main body of the rotating growth vial, or from ½ to ⅓ the lengthof the main body of the rotating growth vial. In other aspects, theremay be concentric rows of raised features disposed on the inner surfaceof the main body of the rotating growth vial arranged horizontally orvertically; and in other aspects, there may be a spiral configuration ofraised features disposed on the inner surface of the main body of therotating growth vial. In alternative aspects, the concentric rows ofraised features or spiral configuration may be disposed upon a post orcenter structure of the rotating growth vial.

In some aspects of the rotating growth vial, the characteristic that ismeasured is optical density. In some aspects the wavelength at which theoptical density is measured is selected by a user. In some aspects thecharacteristic is measured continuously, and in other aspects, thecharacteristic is measured at intervals. In some aspects the rotatinggrowth vial comprises a second light path. In some aspects the rotatingcell growth vial volume is 1-250 ml, 2-100 ml, or 12-35 ml. In someaspects the vial is fabricated from cyclic olefin copolymer (COC),polycarbonate, or polypropylene, and may be fabricated by injectionmolding.

Additionally, some embodiments provide a cell growth device thatcomprises a housing; a motor; a thermal control device; aspectrophotometer; a processor; and a rotating cell growth vialcomprising a vial; a motor assembly configured to be connected to themotor to spin the vial; an electrical connection configured to beelectrically coupled to the thermal control device; a first light paththrough the vial to measure a value of a characteristic of cells in thevial via the spectrophotometer; and a connection to the processor,wherein the processor accepts input from a user, receives from thespectrophotometer the value of the characteristic of the cells,calculates the control actions, and directs the thermal control deviceto adjust the temperature of the vial to grow the cells to a targetvalue at a target time. Also in some aspects, the thermal control devicemay adjust the temperature of the rotating growth vial for, in additionto cell growth, heat shock of the culture, induction oftemperature-sensitive inducible promoters, and for cooling andmaintaining the growth culture at, e.g., 4° C.

In some aspects, the characteristic that is measured is optical density.In some aspects, the wavelength at which the optical density is measuredis part of a script that is programmed into the processor, and in otheraspects the wavelength at which the optical density is measured isspecified by a user. In some aspects, the characteristic is measuredcontinuously, and in other aspects, the characteristic is measured atintervals. In yet other aspects of this embodiment, the characteristicis measured at intervals until a specified value is met, then thecharacteristic is measured continuously. In some aspects the rotatingcell growth vial component of the cell growth device comprises a secondlight path different from the first light path to measure the value ofthe characteristic of cells in the vial via the spectrophotometer.

In some aspects the motor component of the cell growth device isconfigured to hold a constant revolution per minute between 0 and 3000RPM, and in some aspects, the motor comprises directional control. Insome aspects the motor is configured to oscillate the rotating growthvial in opposite directions, e.g., clockwise then counter-clockwiserevolutions. In some aspects the thermal control device component of thecell growth device is a Peltier device.

In some aspects of the cell growth device, the processor is configuredto notify the user when the cells have reached the target value by,e.g., a text such as an SMS (Simple Message System standard) text, emailor other messages delivered to a tablet, smart phone, or other PDA.

In some aspects the processor component is programmed with informationto be used as a blank or control to which the value of thecharacteristic of the cells is compared.

Additionally, a multi-module cell processing system comprising the cellgrowth device is provided. In some aspects, the multi-module cellprocessing system further comprises a cell concentration module, aprotein induction module, a transformation module, an editing module, arecovery module, and/or a storage module.

In some aspects the cell growth device is a first module in a cellgrowth, cell concentration, and cell transformation multi-moduleinstrument.

Also presented herein is a method for using the cell growth devicecomprising: transferring an aliquot of cells into a hermetically-sealed,media-filled cell growth vial; entering a user-preferred targetcharacteristic value and user-preferred time to reach the user-preferredtarget characteristic value into the processor; spinning the vial;measuring the characteristic value of the cells; adjusting thetemperature according to the user-preferred time; determining when thecells reach the user-preferred characteristic value of the cells;cooling the cell growth vial or advancing cells to a next module in anautomated multi-module cell processing system; and notifying a user thatthe cells have reached the characteristic value.

In some aspects, the method further comprises performing the measuring,adjusting, and determining steps until the cells reach theuser-preferred value of the cells. In some aspects, the characteristicmeasured is optical density (where the range measured can be OD 0.1 toOD 10), and in some aspects the user-preferred target characteristicvalue is 2.7 and the target characteristic value is read at OD600.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A depicts one embodiment of a rotating growth vial for use withthe cell growth device described herein. FIG. 1B is a top view of asecond embodiment of a rotating growth vial for use with the cell growthdevice described herein. FIG. 1C is a side perspective view of therotating growth vial of FIG. 1B. FIG. 1D is a side view of the rotatinggrowth vial depicted in FIGS. 1B and 1C. FIG. 1E is a side view of thesecond embodiment of the rotating growth vial shown in FIG. 1D and takenalong line A-A of FIG. 1D.

FIG. 2A illustrates a perspective view of one embodiment of a cellgrowth vial and housing. FIG. 2B depicts a cut-away view of the cellgrowth device from FIG. 2A. FIG. 2C illustrates the cell growth deviceof FIG. 2A coupled to LED, detector, and temperature regulatingcomponents. FIG. 2D illustrates a perspective view of a stand-alone cellgrowth device. FIG. 2E illustrates a cross-section of the stand-alonecell growth device shown in FIG. 2D. FIG. 2F is a flow chart of oneembodiment of a method for using the cell growth device.

FIG. 3A is a model of tangential flow filtration used in the TFF modulepresented herein. FIG. 3B depicts a top view of a lower member of oneembodiment of an exemplary TFF device/module. FIG. 3C depicts a top viewof upper and lower members and a membrane of an exemplary TFF module.FIG. 3D depicts a bottom view of upper and lower members and a membraneof an exemplary TFF module. FIGS. 3E-3H depict various views of anembodiment of a TFF module having fluidically coupled reservoirs forretentate, filtrate, and exchange buffer.

FIGS. 4A and 4B are top perspective and bottom perspective views,respectively, of flow-through electroporation devices (here, there aresix such devices co-joined). FIG. 4C is a top view of one embodiment ofan exemplary flow-through electroporation device. FIG. 4D depicts a topview of a cross section of the electroporation device of FIG. 4C. FIG.4E is a side view cross section of a lower portion of theelectroporation devices of FIGS. 4C and 4D.

FIG. 5 depicts an exemplary automated multi-module cell processinginstrument comprising the growth module.

FIG. 6 is a simplified block diagram of one embodiment of an exemplaryautomated multi-module cell processing instrument that includes a cellgrowth module.

FIG. 7 is a simplified block diagram of yet another embodiment of anexemplary automated multi-module cell processing system that includes acell growth module.

FIG. 8 is a graph demonstrating the effectiveness of a 2-paddle rotatinggrowth vial and cell growth device as described herein for growing anEC23 cell culture vs. a conventional cell shaker.

FIG. 9 is a graph demonstrating the effectiveness of a 3-paddle rotatinggrowth vial and cell growth device as described herein for growing anEC23 cell culture vs. a conventional cell shaker.

FIG. 10 is a graph demonstrating the effectiveness of a 4-paddlerotating growth vial and cell growth device as described herein forgrowing an EC138 cell culture vs. a conventional orbital cell shaker.

FIG. 11 is a graph demonstrating the effectiveness of a 2-paddlerotating growth vial and cell growth device as described herein forgrowing an EC138 cell culture vs. a conventional orbital cell shaker.

FIG. 12 is a graph demonstrating real-time monitoring of growth of anEC138 cell culture to OD₆₀₀ employing the cell growth device asdescribed herein where a 2-paddle rotating growth vial was used.

FIG. 13 is a graph demonstrating real-time monitoring of growth of s288cyeast cell culture OD₆₀₀ employing the cell growth device as describedherein where a 2-paddle rotating growth vial was used.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices and instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York, 1989; and Chang, et al., Guide toElectroporation and Electrofusion, Academic Press, California (1992),all of which are herein incorporated in their entirety by reference forall purposes. Nucleic acid-guided nuclease techniques can be found in,e.g., Genome Editing and Engineering from TALENs and CRISPRs toMolecular Surgery, Appasani and Church (2018); and CRISPR: Methods andProtocols, Lindgren and Charpentier (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The Invention in General

For culture of adherent cells, cells may be disposed on beads,microcarriers, or other type of scaffold suspended in medium. Mostnormal mammalian tissue-derived cells—except those derived from thehematopoietic system—are anchorage dependent and need a surface or cellculture support for normal proliferation. In the rotating growth vialdescribed herein, microcarrier technology is leveraged. Microcarriers ofparticular use typically have a diameter of 100-300 μm and have adensity slightly greater than that of the culture medium (thusfacilitating an easy separation of cells and medium for, e.g., mediumexchange) yet the density must also be sufficiently low to allowcomplete suspension of the carriers at a minimum stirring rate in orderto avoid hydrodynamic damage to the cells. Many different types ofmicrocarriers are available, and different microcarriers are optimizedfor different types of cells. There are positively charged carriers,such as Cytodex 1 (dextran-based, GE Healthcare), DE-52(cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based,Sigma-Aldrich Labware), and HLX 11-170 (polystyrene-based); collagen- orECM- (extracellular matrix) coated carriers, such as Cytodex 3(dextran-based, GE Healthcare) or HyQ-sphere Pro-F 102-4(polystyrene-based, Thermo Scientific); non-charged carriers, likeHyQ-sphere P 102-4 (Thermo Scientific); or macroporous carriers based ongelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GEHealthcare).

For culture of adherent cells, cells may be disposed on beads or anothertype of scaffold suspended in medium. Most normal mammaliantissue-derived cells—except those derived from the hematopoieticsystem—are anchorage dependent and need a surface or cell culturesupport for normal proliferation. In the rotating growth vial describedherein, microcarrier technology is leveraged. Microcarriers ofparticular use typically have a diameter of 100-300 μm and have adensity slightly greater than that of the culture medium (thusfacilitating an easy separation of cells and medium for, e.g., mediumexchange) yet the density must also be sufficiently low to allowcomplete suspension of the carriers at a minimum stirring rate in orderto avoid hydrodynamic damage to the cells. Many different types ofmicrocarriers are available, and different microcarriers are optimizedfor different types of cells. There are positively charged carriers,such as Cytodex 1 (dextran-based, GE Healthcare), DE-52(cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based,Sigma-Aldrich Labware), HLX 11-170 (polystyrene-based); collagen or ECM(extracellular matrix)-coated carriers, such as Cytodex 3(dextran-based, GE Healthcare) or HyQ-sphere Pro-F 102-4(polystyrene-based, Thermo Scientific); non-charged carriers, likeHyQspheres P 102-4 (Thermo Scientific); or macroporous carriers based ongelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GEHealthcare).

Measuring the OD of a cell culture is important for vital proceduressuch as transformation and transfection of cells (collectively referredto generally as “transformation” herein), induction of proteinproduction in cells, and conducting minimal inhibitory concentrationexperiments. Optical density may be determined as the absolute value ofthe logarithm with base 10 of the power transmission factor of anoptical attenuator: OD=−log 10 (power out/power in). OD is opticalattenuation; that is, the sum of absorption, scattering and reflection,thus OD specifies the overall power transmission. As cells grow andbecome denser, the OD of the cell culture increases.

In the cell growth devices described herein, in one embodiment cells areinoculated (pipetted) into a rotating growth vial pre-filled with growthmedia. The rotating growth vial is hermetically sealed with a foil top,and the pipette is used to punch through the foil top. An incubation(e.g., growth) temperature is set by the user or via a pre-programmedprotocol by the processor, typically 30° C., and the processor initiatesrotation of the rotating growth vial by the motor. The cell culture(cells+growth medium) slowly moves vertically up the wall of therotating growth vial due to centrifugal force. The movement of the cellculture up the wall of the vial exposes a large surface area of the cellculture to oxygen in the environment (aeration) to optimize uniformcellular respiration. The cell growth device takes either continuous ODreadings, OD readings at set intervals, or, e.g., takes OD readings atset intervals followed by continuous OD monitoring as the OD gets closerto the target OD. The cell growth device provides a feedback loop thatmonitors cell growth in real time and adjusts the temperature of therotating growth vial in real time to reach the OD at a time specified bya user, as well as terminates the growth of the cell culture at apre-determined OD and cools the cell culture to inhibit further growth.Optionally, the processor of the cell growth device will notify one ormore users when the cells reach the target OD, e.g., via a smart phone,tablet or other device.

Thus, the cell growth device described herein enhances the growth ofcells by providing rotation and aeration of the growing cells andprovides in situ continuous growth rate monitoring within thetemperature-controlled vial. Additionally, the processor may control athermal control device to adjust the temperature of the rotating growthvial for, in addition to cell growth, heat shock of the culture,induction of temperature-sensitive inducible promoters, and for coolingand maintaining the cell culture at, e.g., 4° C.

The Rotating Growth Vial

FIG. 1A shows one embodiment of a rotating growth vial 100 for use withthe cell growth device described herein. The rotating growth vial is anoptically-transparent container having an open end 104 for receivingliquid media and cells, a central vial region 106 that defines theprimary container for growing cells, a tapered-to-constricted region 118defining at least one light path 110, a closed end 116, and a driveengagement mechanism 112. The rotating growth vial has a centrallongitudinal axis 120 around which the vial rotates, and the light path110 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 110 is positioned in the lower constricted portion ofthe tapered-to-constricted region 118. Optionally, some embodiments ofthe rotating growth vial 100 have a second light path 108 in the taperedregion of the tapered-to-constricted region 118. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andis not affected by the rotational speed of the growth vial. The firstlight path 110 is shorter than the second light path 108 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 108 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 112 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 112 such that the rotating growth vial is rotatedin one direction only, and in other embodiments, the rotating growthvial is rotated in a first direction for a first amount of time orperiodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial (and the cell culturecontents) are subjected to an oscillating motion. Further, the choice ofwhether the culture is subjected to oscillation and the periodicitytherefor may be selected by the user. The first amount of time and thesecond amount of time may be the same or may be different. The amount oftime may be 1, 2, 3, 4, 5, or more seconds, or may be 1, 2, 3, 4 or moreminutes. In another embodiment, in an early stage of cell growth therotating growth vial may be oscillated at a first periodicity (e.g.,every 60 seconds), and then a later stage of cell growth the rotatinggrowth vial may be oscillated at a second periodicity (e.g., every onesecond) different from the first periodicity.

The rotating growth vial 100 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 104with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 104 mayoptionally include an extended lip 102 to overlap and engage with thecell growth device (not shown). In automated systems, the rotatinggrowth vial 100 may be tagged with a barcode or other identifying meansthat can be read by a scanner or camera that is part of the automatedsystem (not shown).

The volume of the rotating growth vial 100 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 100 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 100 may range from 1-250 ml, 2-100 ml, from 5-80 ml, 10-50ml, or from 12-35 ml. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 ml growth vial, the volume of the cell culture would be from about1.5 ml to about 26 ml, or from 6 ml to about 18 ml.

The rotating growth vial 100 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polyethylene, polypropylene, polycarbonate, poly(methylmethacrylate (PMMA), polysulfone, polyurethane, and co-polymers of theseand other polymers. Preferred materials include polypropylene,polycarbonate, or polystyrene. In some embodiments, the rotating growthvial is inexpensively fabricated by, e.g., injection molding orextrusion.

FIGS. 1B-1D show a second embodiment of a rotating growth vial for usewith the cell growth device. FIG. 1B is a top view of the secondembodiment of a rotating growth vial 100. As in FIG. 1A, the rotatinggrowth vial is an optically transparent container having an open end 104for receiving liquid media and cells. Also shown is extended lip 102configured to overlap and engage with the cell growth device (not shown)and to allow a grip for a user to insert and remove the rotating growthvial from the cell growth device. In this second embodiment of therotating growth vial, two inner “paddles” 150 can be seen extending fromthe inner wall of the rotating growth vial toward the center of thecentral vial region 106.

FIG. 1C is a side perspective view of the second embodiment of arotating growth vial shown in FIG. 1B. As in FIG. 1A, the rotatinggrowth vial is a transparent container having an open end 104 forreceiving liquid media and cells, a central vial region 106 that definesthe primary container for growing cells, a tapered-to-constricted region118 defining at least one light path 110, and a drive engagementmechanism 112. The light path 110 is positioned in the lower constrictedportion of the tapered-to-constricted region 118 and is positioned in aregion of the rotating growth vial that is constantly filled with thecell culture (cells+growth media) and is not affected by the rotationalspeed of the growth vial. The drive engagement mechanism 112 engageswith a motor (not shown) to rotate the vial. Also shown in FIG. 1C arepaddles 150 extending from the inner wall of the rotating growth vialtoward the center of the central vial region 106.

FIG. 1D is a side view of the rotating growth vial of FIGS. 1B and 1Cwhich more clearly show the paddles or interior features of the rotatinggrowth vial. FIG. 1D shows a rotating growth vial with a central vialregion 106 that defines the primary container for growing cells, atapered-to-constricted region 118 defining at least one light path 110,a closed end 116, and a drive engagement mechanism 112. The rotatinggrowth vial has a central longitudinal axis A-A around which the vialrotates. The light path 110 is generally perpendicular to thelongitudinal axis of the vial and is positioned in the lower constrictedportion of the tapered-to-constricted region 118. The drive engagementmechanism 112 engages with a motor (not shown) to rotate the vial. Alsoshown in FIG. 1D are two paddles 150 extending from the inner wall ofthe rotating growth vial 100 toward the center of the central vialregion 106. FIG. 1D shows that the widths and lengths of paddles 150 canvary; for example, the width may be short as in A, longer as in B, andeven longer as in C. Preferably, the width of both paddles 150 is thesame.

FIG. 1E is a side view of the second embodiment of the rotating growthvial 100 shown in FIG. 1D and taken along line A-A of FIG. 1D andlooking perpendicularly at paddle 150 the side of which is shown in FIG.1D. Shown is central vial region 106 that defines the primary containerfor growing cells, a tapered-to-constricted region 118 defining at leastone light path 110, a closed end 116, and a drive engagement mechanism112. Also shown is extended lip 102 configured to overlap and engagewith the cell growth device (not shown) and to allow a grip for a userto insert and remove the rotating growth vial from the cell growthdevice.

Like the rotating growth vial depicted in FIG. 1A, the paddle embodimentof the rotating growth vial 100 may be reusable, or preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial 100 is consumable and is presented to the user pre-filledwith growth medium, where the vial is hermetically sealed at the openend 104 with a foil seal. A medium-filled rotating growth vial packagedin such a manner may be part of a kit for use with a stand-alone cellgrowth device or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip (or needle) to punch through the foil seal of the vial. Open end 104may optionally include an extended lip 102 to overlap and engage withthe cell growth device (not shown). In automated systems, the rotatinggrowth vial 100 may be tagged with a barcode or other identifying meansthat can be read by a scanner or camera that is part of the automatedsystem (not shown).

The volume of the paddle embodiment of the rotating growth vial 100 andthe volume of the cell culture (including growth medium) may varygreatly, but the volume of the paddle embodiment of the rotating growthvial 100, like a growth vial without interior features, must be largeenough for the cell culture in the growth vial to get proper aerationwhile the vial is rotating as described above.

Although the paddle embodiment of the rotating growth vial 100 depictedin FIGS. 1B-1D shows two paddles, the rotating growth vial may comprise2, 3, 4, 5, 6 or more paddles, and up to 20 paddles. The number ofpaddles will depend upon, e.g., the size or volume of the rotatinggrowth vial 100. The paddles may be arranged symmetrically as singlepaddles extending from the inner wall of the vial into the interior ofthe vial, or the paddles may be symmetrically arranged in groups of 2,3, 4 or more paddles in a group (for example, a pair of paddles oppositeanother pair of paddles) extending from the inner wall of the vial intothe interior of the vial. In another embodiment, the paddles may extendfrom the middle of the rotating growth vial out toward the inner wall ofthe rotating growth vial, from, e.g., a post or other support structurein the interior of the rotating growth vial.

Similarly, the size and configuration of the paddles may vary greatlydepending on the size or volume of the rotating growth vial 100. Forexample, FIG. 1D depicts paddles 150 of varying width A, B and C.However, these widths are exemplary only. Because the actual measurementof the paddles varies with the size or volume of the rotating growthvial 100, it is perhaps more instructive to describe the dimensions ofthe paddles with respect to the rotating growth vial 100. For example,in FIG. 1D, width A is approximately 1/12 the diameter of the vial,width B is approximately 1/7 the diameter of the vial, and width C isapproximately ¼ the diameter of the vial. Thus, the width of the paddlesmay range from 1/20 to just under ½ the diameter of the rotating growthvial, or from 1/15 to ⅓ the diameter of the rotating growth vial, orfrom 1/10 to ¼ the diameter of the rotating growth vial. The actualmeasurement of the length of the paddles also will vary with the size orvolume of the rotating growth vial 100, and may extend only through thecentral vial region 106 of the rotating growth vial 100, or may alsoextend into the tapered portion of the tapered-to-constricted region118. The length of the paddles may range from ⅘ to ¼ the length of thecentral vial region 106 of the rotating growth vial 100, or from ¾ to ⅓the length of the central vial region 106 of the rotating growth vial100, or from ½ to ⅓ the length of the central vial region of therotating growth vial 100.

The configuration or shape of the paddles or interior features may varyas well and may essentially embody any shape that may be configured intoa paddle or other feature. For example, the paddles may extendperpendicularly from, e.g., the inner wall of the rotating growth vial100, or the paddles may be curved, e.g., to the left or right ofperpendicular. As shown in FIG. 1D, the end of the paddles proximal tothe open end 104 of the rotating growth vial 100 may be tapered. In FIG.1D, the end of the paddles are tapered from high to low as the paddleextends from the inner wall toward the center of the rotating growthvial 100; however, embodiments where the paddles are tapered from low tohigh as the paddle extends from the inner wall to the center of therotating growth vial 100 are also contemplated, as are untamperedpaddles. Also shown in FIG. 1D, the paddles extend from the central vialregion 106 into tapered-to-constricted region 118, although this is onlyone option for configuring the paddles.

In another embodiment, there may be concentric rows of raised regionsdisposed on the inner surface of the central vial region 106 of therotating growth vial 100 arranged horizontally or vertically (embodimentnot shown); and in another embodiment, there may be a spiralconfiguration of raised regions disposed on the inner surface of thecentral vial region 106 of the rotating growth vial 100. In alternativeaspects, the concentric rows of raised regions or spiral configurationmay be disposed upon a post or center structure of the rotating growthvial 100.

The paddles 150 of the rotating growth vial 100—like the rotating growthvial itself—are preferably fabricated from a bio-compatible transparentmaterial, and the material from which the paddles are fabricated shouldbe able to be cooled to about 4° C. or lower and heated to about 55° C.or higher to accommodate both temperature-based cell assays andlong-term storage at low temperatures. Exemplary materials from which tofabricate the paddles—like the rotating growth vial 100—are listedabove. Preferably, the rotating growth vial and paddles are fabricatedin one piece by, e.g., injection molding or extrusion.

The Cell Growth Device

FIG. 2A is a perspective view of one embodiment of a cell growth device200. FIG. 2B depicts a cut-away view of the cell growth device 200 fromFIG. 2A. In both figures, the rotating growth vial 100 is seenpositioned inside a main housing 206 with the extended lip 102 of therotating growth vial 100 extending above the main housing 206.Additionally, end housings 222, a lower housing 202 and flanges 204 areindicated in both figures. Flanges 204 are used to attach the cellgrowth device 200 to heating/cooling means or other structure (notshown). FIG. 2B depicts additional detail. In FIG. 2B, upper bearing 212and lower bearing 210 are shown positioned in main housing 206. Upperbearing 212 and lower bearing 210 support the vertical load of rotatinggrowth vial 100. Lower housing 202 contains the drive motor 208. Thecell growth device 200 of FIG. 2B comprises two light paths: a primarylight path 214, and a secondary light path 220. Light path 214corresponds to light path 110 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 100, andlight path 220 corresponds to light path 108 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth vial. Lightpaths 110 and 108 are not shown in FIG. 2B but may be seen in, e.g.,FIG. 1A. In addition to light paths 214 and 220, there is an emissionboard 218 to illuminate the light path(s), and detector board 216 todetect the light after the light travels through the cell culture liquidin the rotating growth vial 100.

The motor 208 used to rotate the rotating growth vial 200 in someembodiments is a brushless DC type drive motor with built-in drivecontrols that can be set to hold a constant revolution per minute (RPM)between 0 and about 3000 RPM. Alternatively, other motor types such as astepper, servo, brushed DC, and the like can be used. Optionally, themotor 208 may also have direction control to allow reversing of therotational direction, and a tachometer to sense and report actual RPM.The motor is controlled by a processor (not shown) according to, e.g.,standard protocols programmed into the processor and/or user input, andthe motor may be configured to vary RPM to cause axial precession of thecell culture thereby enhancing mixing, e.g., to prevent cellaggregation, increase aeration, and optimize cellular respiration.

Main housing 206, end housings 222 and lower housing 202 of the cellgrowth device 200 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 100 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 200 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 200 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processorof the cell growth device 200—may be programmed to use wavelength valuesfor blanks commensurate with the growth media typically used in cellculture (whether, e.g., mammalian cells, bacterial cells, animal cells,yeast cells, etc.). Alternatively, a second spectrophotometer and vesselmay be included in the cell growth device 200, where the secondspectrophotometer is used to read a blank at designated intervals.

FIG. 2C illustrates a cell growth device 200 as part of an assemblycomprising the cell growth device 200 of FIG. 2A coupled to light source290, detector 292, and thermal components 294. The rotating growth vial100 is inserted into the cell growth device. Components of the lightsource 290 and detector 292 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 202 that houses the motor that rotatesthe rotating growth vial 100 is illustrated, as is one of the flanges204 that secures the cell growth device 200 to the assembly. Also, thethermal components 294 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 200 to the thermal component 294 via the flange 204 on the baseof the lower housing 202. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 200 is controlled to approximately +/−0.5° C.

FIG. 2D is a perspective view and FIG. 2E is a cross-section of astand-alone cell growth device 300 that has been built and tested. FIG.2D shows the rotating growth vial 100 inserted into a housing 304 alongwith an LCD touch screen 302. The cell growth device 300 shown in FIG.2D has the following dimensions: H=146 mm, L=225 mm, and D=153 mm;although these dimensions are, of course, exemplary. This embodiment ofthe device weighed approximately 4.05 kg, required 40 watts of power forthe heating, cooling, and fan; 5 watts of power for the rotationalmotor; 5 watts of power for the light emitting diode and photodetector;and 5 watts of power for the single board computer anddigital-analog-input-output boards (all described below). The exteriorof the cell growth device 300 consists in this embodiment of a set ofsheet metal case halves on a base that holds all the componentsnecessary to run the module. A 120 VAC International ElectrotechnicalCommission (IEC) standard power cord (not shown) plugged into a walloutlet is all that is needed to power the unit.

In this embodiment, a rear-mounted power entry module 316 contains thesafety fuses and the on-off switch, which when switched on powers theinternal AC and DC power supplies (not shown) activating a built-insingle board computer which runs, e.g., a Windows 10 operating system.The cell growth module in this embodiment has no wired physical externalcommunications connections, but optional communications can be accessedvia the built-in Wi-Fi interface. The cell growth module can beconfigured to operate remotely or by the user using the attached 7″ LCDtouch screen 302. Security and access controls optionally may be enabledthat, e.g., require logons to activate the programs and to run thegrowth functions. Likewise, access via remote laptops or desktops isconfigurable—and if allowed on the network—parameters such as growthdata, graphs, and sequence progress can be sent out at intervals viaemail or text messages. Any updates to programs or operating systems canbe “pushed” out remotely as well, again depending on network securitysettings.

FIG. 2E is a cross-section of the stand-alone cell growth device shownin FIG. 2D, illustrating internal components. The major components are athermoelectric cooler control 306 and a thermoelectric cooler 320, alsoknown as a Peltier device, capable of heating or cooling at a 40-wattcapacity. Also seen are the control electronics 308, which includes asingle board computer as well as analog and digital boards attached tothe main housing that contain the optical systems for making the ODmeasurements. A rotating growth vial housing 318 contains bearings (notshown), a drive motor 208 (e.g., a brushless DC type motor), and a drivecoupling 312 which manages the thermal environment and mechanicalrotation of the rotating growth vial 100. Bearings (not shown) at thetop and bottom of the rotating growth vial 100 support the vertical axisof the rotating growth vial and the motor drive coupling 312 engages therotating growth vial to allow the drive motor 208 to spin the rotatinggrowth vial 100. A through-hole 330 is seen that allows the, e.g., 600nm LED 310 located on the digital board control electronics 308 toilluminate through-hole 330 in the rotating growth vial housing 318,through the lower constricted section of the rotating growth vial 100,continuing through the through-hole opening 330 on the other side of thehousing, and ending at analog photodiode detection board 314. The lowestsection of the main housing contains the drive motor 208, in this case abrushless DC type that has built in drive controls that set and hold aconstant rate of speed measured in revolutions per minute (RPM) between0 and 3000 RPM. There is also direction control to allow programmedreversing of the rotational direction, and a tachometer output to senseactual RPM.

Thermal control is accomplished by, e.g., attachment of the housing to aPeltier device, commonly known as a thermoelectric cooler 320. Peltierdevices are capable of ‘pumping’ heat to either side of their junctions,either cooling a surface or heating a surface depending on the directionof current flow. A thermistor is used to measure the temperature of themain housing and then through a standard electronicProportional-Integral-Derivative (PID) controller (e.g., the temperaturecontrol board is mounted in the control electronics 308) the programmedset temperature is controlled to an accuracy of approximately +/−0.5° C.Any difference from the housing temperature (at the thermistor) and theliquid media temperature, measured inside the rotating growth vial, ispre-calibrated as an offset temperature and stored in the settings ofthe software control program.

Measurements of optical densities (OD) at programmed time intervals areaccomplished using a 600 nm Light Emitting Diode (LED) 310 that has beencolumnated through an optic into the lower constricted portion of therotating growth vial which contains the cells of interest. The lightcontinues through a collection optic to the detection system whichconsists of a (digital) gain-controlled silicone photodiode 314.Generally, optical density is normally shown as the absolute value ofthe logarithm with base 10 of the power transmission factors of anoptical attenuator: OD=−log 10 (Power out/Power in). Since OD is themeasure of optical attenuation—that is, the sum of absorption,scattering, and reflection—the cell growth device OD measurement recordsthe overall power transmission, so as the cells grow and become denserin population, the OD (the loss of signal) increases. The OD system ispre-calibrated against OD standards with these values stored in anon-board memory accessible by the measurement program.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial by piercing though the foil seal. Theprogrammed software of the cell growth device sets the controltemperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial to expose a large surface area of the mixtureto a normal oxygen environment. The growth monitoring system takeseither continuous readings of the OD or OD measurements at pre-set orpre-programmed time intervals. These measurements are stored in internalmemory and if requested the software plots the measurements versus timeto display a growth curve. If enhanced mixing is required, e.g., tooptimize growth conditions, the speed of the vial rotation can be variedto cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 200 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device has been described inthe context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.For example, spectroscopy using visible, UV, or near infrared (NIR)light allows monitoring the concentration of nutrients and/or wastes inthe cell culture. Additionally, spectroscopic measurements may be usedto quantify multiple chemical species simultaneously. Nonsymmetricchemical species may be quantified by identification of characteristicabsorbance features in the NIR. Conversely, symmetric chemical speciescan be readily quantified using Raman spectroscopy. Many criticalmetabolites, such as glucose, glutamine, ammonia, and lactate havedistinct spectral features in the IR, such that they may be easilyquantified. The amount and frequencies of light absorbed by the samplecan be correlated to the type and concentration of chemical speciespresent in the sample. Each of these measurement types provides specificadvantages. FT-NIR provides the greatest light penetration depth and canbe used for thicker samples. FT-mid-IR (MIR) provides information thatis more easily discernible as being specific for certain analytes asthese wavelengths are closer to the fundamental IR absorptions. FT-Ramanis advantageous when interference due to water is to be minimized. Otherspectral properties can be measured via, e.g., dielectric impedancespectroscopy, visible fluorescence, fluorescence polarization, orluminescence. Additionally, the cell growth device may includeadditional sensors for measuring, e.g., dissolved oxygen, carbondioxide, pH, conductivity, and the like.

FIG. 2F is a block diagram of one method 3000 for using the cell growthdevice including measuring OD and providing feedback. In a first step ofthe method, a user transfers an aliquot of cells into a media-filledcell growth vial 3002. The user then specifies via input into aprocessor a target OD and preferred time that the cell culture reach thetarget OD 3004. The user can manually enter these parameters, the usermay, e.g., choose from a menu of established protocols and parameters,or, in an automated cell processing system, there may be the option ofusing a barcode or other tag on the rotating growth vial that specifiesthe protocols and parameters (e.g., the medium contained within thevial, the cell type, the wavelength to read OD, and the desired opticaldensity endpoint) where the barcode is detected by the processor thatcontrols the cell growth device.

At step 3006, the rotation of the rotating cell vial is initiated at aspecific temperature. Again, a user may manually specify the temperatureand rotation rate of the vial, the user may choose from a menu ofestablished protocols and parameters, or there may be an option of usinga barcode or other tag that specifies the protocols and parameters inadvance. At step 3008, the OD of the cell culture in the vial ismeasured. As described above, OD may be measured continuously, may bemeasured at specific time intervals, or a combination of the two can beused. The cell growth module then adjusts the temperature of therotating growth vial (and the cell culture) according to the target timerequested by the user 3010. The steps of measuring OD 3008 and adjustingthe temperature of the cell culture 3010 continue until the cell culturereaches the target OD 3012. At this point, the processor may send acommand to the thermal control device to cool the growth vial to, e.g.,4° C. or to freeze the cells. Alternatively, if the cell growth deviceis part of a system—e.g., one module in a multi-module cell processingsystem—the processor may advance the cells to a next module 3014. Inaddition, at step 3016, the processor may notify the user that the cellshave reached a target OD, for example, through an application on theuser's cell phone or other digital assistant.

Use of the Cell Growth Device in an Automated Multi-Module CellProcessing Instrument

As mentioned above, the cell growth device may be used as a stand-aloneinstrument such as a benchtop instrument, or as a module in an automatedmulti-module cell processing instrument. One such instrument is aninstrument that combines a cell growth module, a cellconcentration/buffer exchange module (“cell concentration module”), anda transformation/transfection module (“transformation module”), where aliquid handling system transfers liquids between reagent reservoirs andthe three modules automatically without human intervention. FIGS. 3A-3Hdepict variations of one embodiment of a cell concentration/bufferexchange module that utilizes tangential flow filtration.

The cell concentration module described herein operates using tangentialflow filtration (TFF), also known as cross-flow filtration, in which themajority of the feed flows tangentially over the surface of the filterthereby reducing cake (retentate) formation as compared to dead-endfiltration, in which the feed flows into the filter. Secondary flowsrelative to the main feed are also exploited to generate shear forcesthat prevent filter cake formation and membrane fouling thus maximizingparticle recovery, as described below.

The TFF device described herein was designed to take into account twoprimary design considerations. First, the geometry of the TFF deviceleads to filtering the cell culture over a large surface area so as tominimize processing time. Second, the design of the TFF device isconfigured to minimize filter fouling. FIG. 3A is a general model 450 oftangential flow filtration. The TFF device operates using tangentialflow filtration, also known as cross-flow filtration. FIG. 3A showscells flowing over a membrane 424, where the feed flow of the cells 452in medium or buffer is parallel to the membrane 424. TFF is differentfrom dead-end filtration where both the feed flow and the pressure dropare perpendicular to a membrane or filter.

FIG. 3B depicts a top view of a lower member 420 of one embodiment of anexemplary TFF device/module providing tangential flow filtration. As canbe seen in FIG. 3B, the lower member 420 of the TFF device modulecomprises a channel structure 416 comprising a flow channel throughwhich a cell culture is flowed. The channel structure 416 comprises asingle flow channel 402 that is horizontally bifurcated by a membrane(not shown) through which buffer or medium may flow, but cells cannot.(Note, that the flow channel generally is designated 402, the portion ofthe flow channel in the upper member 422 of the TFF device is designated402 a, and the portion of the flow channel in the lower member 420 ofthe TFF device is designated 402 b.) This particular embodimentcomprises a channel configuration 414, e.g., an undulating serpentinegeometry (i.e., the small “wiggles” in the flow channel 402) and aserpentine “zig-zag” pattern where the flow channel 402 b crisscrossesthe lower member 420 of the TFF device from one end at the left of thedevice to the other end at the right of the device. The serpentinepattern allows for filtration over a high surface area relative to thedevice size and total channel volume, while the undulating contributioncreates a secondary inertial flow to enable effective membraneregeneration preventing membrane fouling. Although an undulatinggeometry and serpentine pattern are exemplified here, other channelconfigurations 414 may be used as long as the flow channel 402 can bebifurcated by a membrane, and, as discussed below, as long as thechannel configuration 414 provides for cell flow through the TFF modulein alternating directions. Portals 404 and 406 are part of channelstructure 416 by operation of the cells passing through flow channel402. Generally, portals 404 collect cells passing through the flowchannel 402 on one side of a membrane (not shown) (the “retentate”), andportals 406 collect the medium (“filtrate” or “permeate”) passingthrough the flow channel 402 on the opposite side of the membrane (notshown). In this embodiment, recesses 408 accommodate screws or otherfasteners (not shown) that allow the components of the TFF device to besecured to one another.

The length 410 and width 412 of the channel structure 416 may varydepending on the volume of the cell culture to be grown and the opticaldensity of the cell culture to be concentrated. The length 410 of thechannel structure 416 typically is from 1 mm to 300 mm, or from 50 mm to250 mm, or from 60 mm to 200 mm, or from 70 mm to 150 mm, or from 80 mmto 100 mm. The width of the channel structure 416 typically is from 1 mmto 120 mm, or from 20 mm to 100 mm, or from 30 mm to 80 mm, or from 40mm to 70 mm, or from 50 mm to 60 mm. The cross-section configuration ofthe flow channel 402 may be round, elliptical, oval, square,rectangular, trapezoidal, or irregular. If square, rectangular, oranother shape with generally straight sides, the cross section may befrom about 10 μm to 1000 μm wide, or from 200 μm to 800 μm wide, or from300 μm to 700 μm wide, or from 400 μm to 600 μm wide; and from about 10μm to 1000 μm high, or from 200 μm to 800 μm high, or from 300 μm to 700μm high, or from 400 μm to 600 μm high. If the cross section of the flowchannel 102 is generally round, oval or elliptical, the radius of thechannel may be from about 50 μm to 1000 μm in hydraulic radius, or from5 μm to 800 μm in hydraulic radius, or from 200 μm to 700 μm inhydraulic radius, or from 300 μm to 600 μm wide in hydraulic radius, orfrom about 200 to 500 μm in hydraulic radius.

When looking at the top view of lower member 420 of the TFFdevice/module of FIG. 3B, note that there are two retentate portals 404and two filtrate portals 406, where there is one of each type of portalat both ends (e.g., the narrow edge) of the TFF device/module 400. Inother embodiments, retentate and filtrate portals can be on the samesurface of the same member (e.g., upper 422 or lower member 420), orthey can be arranged on the side surfaces of the assembly. Unlike othertangential flow filtration devices that operate continuously, the TFFdevice/module described herein uses an alternating method forconcentrating cells. The overall work flow for cell concentration usingthe TFF device/module involves flowing a cell culture or cell sampletangentially through the channel structure 416. The membrane bifurcatingthe flow channels 402 retains the cells on one side of the membrane andallows unwanted medium or buffer to flow across the membrane into thefiltrate side (e.g, lower member 420) of the device. In this process, afixed volume of cells in medium or buffer is driven through the deviceuntil the cell sample is collected into one of the retentate portals404, and the medium/buffer that has passed through the membrane iscollected through one or both of the filtrate portals 406. All types ofprokaryotic and eukaryotic cells—both adherent and non-adherentcells—can be concentrated in the TFF device. Adherent cells may be grownon beads or other cell scaffolds suspended in medium in the rotatinggrowth vial, then passed through the TFF device.

In the cell concentration process, passing the cell sample through theTFF device and collecting the cells in one of the retentate portals 404while collecting the medium in one of the filtrate portals 406 isconsidered “one pass” of the cell sample. The transfer between retentatereservoirs “flips” the culture. The retentate and filtrate portalscollecting the cells and medium, respectively, for a given pass resideon the same end of TFF device/module 400 with fluidic connectionsarranged so that there are two distinct flow layers (not shown) for theretentate and filtrate sides, but if the retentate portal 404 resides onthe upper member 422 of the TFF device/module 400 (that is, the cellsare driven through the flow channel 402 a (not shown) above the membraneand the filtrate (medium) passes to the portion of the flow channel 402b below the membrane), the filtrate portal 406 will reside on the lowermember of device/module 400 and vice versa (that is, if the cell sampleis driven through the flow channel 402 b below the membrane, thefiltrate (medium) passes to the portion of the flow channel 402 a abovethe membrane). This configuration can be seen more clearly in FIGS.3C-3D, where the retentate flows 460 from the retentate portals 404 andthe filtrate flows 470 from the filtrate portals 406.

At the conclusion of a “pass”, the cell sample is collected by passingthrough the retentate portal 404 and into the retentate reservoir (notshown). To initiate another “pass”, the cell sample is passed againthrough the TFF device, this time in a flow direction that is reversedfrom the first pass. The cell sample is collected by passing through theretentate portal 404 and into retentate reservoir (not shown) on theopposite end of the device/module from the retentate portal 404 that wasused to collect cells during the first pass. Likewise, the medium/bufferthat passes through the membrane on the second pass is collected throughthe filtrate portal 406 on the opposite end of the device/module fromthe filtrate portal 406 that was used to collect the filtrate during thefirst pass, or through both portals. This alternating process of passingthe retentate (the concentrated cell sample) through the device/moduleis repeated until the cells have been concentrated to a desired volume,and both filtrate portals can be open during the passes to reduceoperating time. In addition, buffer exchange may be effected by adding adesired buffer (or fresh medium) to the cell sample in the retentatereservoir, before initiating another “pass”, and repeating this processuntil the old medium or buffer is diluted and filtered out and the cellsreside in fresh medium or buffer. Note that buffer exchange and cellconcentration may (and typically do) take place simultaneously.

FIG. 3C depicts a top view of upper (422) and lower (420) members of anexemplary TFF module 400. Again, portals 404 and 406 are seen. As notedabove, recesses—such as the recesses 408 seen in FIG. 3B—provide a meansto secure the components (upper member 422, lower member 420, andmembrane 424) of the TFF device/membrane 400 to one another duringoperation via, e.g., screws or other like fasteners. However, inalternative embodiments, an adhesive—such as a pressure sensitiveadhesive—or ultrasonic welding, or solvent bonding, may be used tocouple the upper member 422, lower member 420, and membrane 424together. Indeed, one of ordinary skill in the art given the guidance ofthe present disclosure can find yet other configurations for couplingthe components of the TFF device 400, such as e.g., clamps; matedfittings disposed on the upper (422) and lower (420) members;combination of adhesives, welding, solvent bonding, and mated fittings;and other such fasteners and couplings.

Note that in FIG. 3C there is one retentate portal 404 and one filtrateportal 406 on each “end” (e.g., the narrow edges) of the TFFdevice/module 400. The retentate 404 and filtrate 406 portals on theleft side of the TFF device/module 400 will collect cells (flow path at460) and medium (flow path at 470), respectively, for the same pass.Likewise, the retentate 404 and filtrate 406 portals on the right sideof the TFF device/module 400 will collect cells (flow path at 460) andmedium (flow path at 470), respectively, for the same pass. In thisembodiment, the retentate is collected from portals 404 on the topsurface of the TFF device, and filtrate is collected from portals 406 onthe bottom surface of the device. The cells are maintained in the TFFflow channel 402 a above the membrane 424, while the filtrate (medium)flows through membrane 424 and then through filtrate portals 406; thus,the top/retentate portals 404 and bottom/filtrate portals 406configuration is practical. It should be recognized, however, that otherconfigurations of retentate 404 and filtrate 406 portals may beimplemented such as positioning both the retentate 404 and filtrate 406portals on the side surface (as opposed to the top and bottom surfaces)of the TFF device 400. In FIG. 3C, the flow channel 402 b can be seen onthe lower member 420 of the TFF device 400. However, in otherembodiments, retentate 404 and filtrate 406 portals can reside on thesame surface of the TFF device.

Also seen in FIG. 3C is membrane or filter 424. Filters or membranesappropriate for use in the TFF device/module 400 are those that aresolvent resistant, are contamination free during filtration, and areable to retain the types and sizes of cells of interest. For example, inorder to retain small cell types such as bacterial cells, pore sizes canbe as low as 0.2 μm, however for other cell types, the pore sizes can beas high as 5 μm. Indeed, the pore sizes useful in the TFF device/module400 include filters 424 with sizes from 0.20 μm, 0.21 μm, 0.22 μm, 0.23μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. The filters 424 may befabricated from any suitable non-reactive material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, glass fiber, or metal substratesas in the case of laser or electrochemical etching. The TFF device 400shown in FIGS. 3C and 3D does not show a seat in the upper 422 and lower420 members where the filter 424 can be seated or secured (for example,a seat half the thickness of the filter 424 in each of upper 422 andlower 420 members); however, such a seat is contemplated in someembodiments.

FIG. 3D depicts a bottom view of upper and lower components of theexemplary TFF module shown in FIG. 3C. FIG. 3D depicts a bottom view ofupper (422) and lower (420) members of an exemplary TFF module 400.Again portals 404 and 406 are seen. Note again that there is oneretentate portal 404 and one filtrate portal 406 on each end of theupper member 422 and lower member 420 of the TFF device/module 400. Onthe left side of the TFF device 400, the retentate portals 404 willcollect cells (flow path at 460) and the filtrate portals 406 willcollect medium (flow path at 470), respectively, for the same pass.Likewise, on the right side of the TFF device 400, the retentate portals404 will collect cells (flow path at 460) and the filtrate portals 406will collect medium (flow path at 470), respectively, for the same pass.In FIG. 3D, the flow channel 402 a can be seen on the upper member 422of the TFF device 400. Thus, looking at FIGS. 3C and 3D, note that thereis a flow channel 402 in both the upper member 422 (flow channel 402 a)and lower member 422 (flow channel 402 b) with a membrane 424 betweenthe upper 422 and lower 420 members. The flow channels 402 a and 402 bof the upper 422 and lower 420 members mate to create the flow channel402 with the membrane 424 positioned horizontally between the upper andlower members of the TFF device/module thereby bifurcating the flowchannel 402.

Buffer exchange during cell concentration and/or rendering the cellscompetent is performed on the TFF device/module 400 by adding a desiredbuffer to the cells concentrated to a desired volume; for example, afterthe cells have been concentrated at least 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold,200-fold or more. A desired exchange medium or exchange buffer is addedto the cells either by addition to the retentate reservoir (not shown)or through the membrane 424 from the filtrate side (e.g., to cells inretentate reservoir) and the process of passing the cells through theTFF device 400 is repeated until the cells have been concentrated to adesired volume in the exchange medium or buffer. This process can berepeated any number of desired times so as to achieve a desired level ofexchange of the buffer and a desired volume of cells. As described inthe Example I, the exchange buffer may comprise, e.g., glycerol orsorbitol thereby rendering the cells competent for transformation inaddition to decreasing the overall volume of the cell sample.

The TFF device 400 may be fabricated from any robust material in whichflow channels may be milled including stainless steel, silicon, glass,aluminum, or plastics including cyclic-olefin copolymer (COC),cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module 400 is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module 400 is thermally-conductive so thatthe cell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device 400 is formed by precisionmechanical machining, laser machining, electro discharge machining (formetal devices); wet or dry etching (for silicon devices); dry or wetetching, powder or sandblasting, photostructuring (for glass devices);or thermoforming, injection molding, hot embossing, or laser machining(for plastic devices) using the materials mentioned above that areamenable to these mass production techniques.

FIG. 3E depicts an exploded perspective view of one exemplary embodimentof a TFF module having fluidically coupled reservoirs for retentate,filtrate, and exchange buffer. In this configuration, 470 is the top orcover of the TFF device, having three ports 466, where there is apipette tip 468 disposed in the left-most port 466. The top 470 of theTFF device is, in operation, coupled with a combined reservoir and uppermember structure 464. Combined reservoir and upper member structure 464comprises a top surface that, in operation, is adjacent the top or cover470 of the TFF device, a bottom surface which comprises the upper member422 of the TFF device, where the upper member 422 of the TFF devicedefines the upper portion of the tangential flow channel (not shown).Combined reservoir and upper member structure 464 comprises tworetentate reservoirs 472 and buffer or medium reservoir 474. Theretentate reservoirs 472 are fluidically coupled to the upper portion ofthe flow channel, and the buffer or medium reservoir 474 is fluidicallycoupled to the retentate reservoirs 472. Also seen in this exploded viewof the TFF device is lower member 420 which, as described previously,comprises on its top surface the lower portion of the tangential flowchannel 402 b (seen on the top surface of lower member 420), where theupper and lower portions of the flow channel 402 of the upper member 422and lower member 420, respectively, when coupled mate to form a singleflow channel 402 (the membrane that is interposed between the uppermember 422 and lower member 420 in operation is not shown). Beneathlower member 420 is gasket 460, which in operation is interposed betweenlower member 420 and a filtrate (or permeate) reservoir 462. Inoperation, top 470, combined reservoir and upper member structure 464,membrane (not shown), lower member 420, gasket 460, and filtratereservoir 462 are coupled and secured together to be fluid- andair-tight. In FIG. 3E, fasteners are shown that can be used to couplethe various structures (top 470, combined reservoir and upper memberstructure 464, membrane (not shown), lower member 420, gasket 460, andfiltrate reservoir 462) together. However, as an alternative to screwsor other like fasteners, the various structures of the TFF device can becoupled using an adhesive, such as a pressure sensitive adhesive;ultrasonic welding; or solvent bonding. Further, a combination offasteners, adhesives, and/or welding types may be employed to couple thevarious structures of the TFF device. One of ordinary skill in the artgiven the guidance of the present disclosure could find yet otherconfigurations for coupling the components of the TFF device, such as,e.g., clamps, mated fittings, and other such fasteners.

FIG. 3F depicts combined reservoir and upper member structure 464,comprising two retentate reservoirs 472 and buffer or medium reservoir474, as well as upper member 422, which is disposed on the bottom ofcombined reservoir and upper member structure 464. Upper member 422 ofthe TFF device defines the upper portion of the tangential flow channel(not shown) disposed on the bottom surface of the combined reservoir andupper member structure 464. FIG. 3G is a top-down view of the uppersurface 478 of combined reservoir and upper member structure 464,depicting the top 480 of retentate reservoirs 472 and the top 482 ofbuffer or medium reservoir 474. The retentate reservoirs 472 arefluidically coupled to the upper portion of the flow channel (notshown), and the buffer or medium reservoir 474 is fluidically coupled tothe retentate reservoirs 472. FIG. 3H is a bottom-up view of the lowersurface 490 of combined reservoir and upper member structure 464,showing the upper member 422 with the upper portion of the tangentialflow channel 402 a disposed on the bottom surface of upper member 422.The flow channel 402 a disposed on the bottom surface of upper member422 in operation is mated to the bottom portion of the tangential flowchannel 402 b disposed on the top surface of the lower member 420 (notshown in this view, but see FIG. 3E), where the upper and lower portionsof the flow channels 402 a and 402 b, respectively, mate to form asingle flow channel 402 with a membrane or filter (not shown) interposedbetween the upper 402 a and lower 402 b portions of the flow channel.

Turning to the third module of the three-module cell growth, cellconcentration, and cell transformation instrument, FIGS. 4A-4E depictvariations on one embodiment of a cell transformation module (in thiscase, a flow-through electroporation device) that may be included in acell growth/concentration/transformation instrument. FIGS. 4A and 4B aretop perspective and bottom perspective views, respectively, of sixco-joined flow-through electroporation devices 4150. FIG. 4A depicts sixflow-through electroporation units 4150 arranged on a single substrate4156. Each of the six flow-through electroporation units 4150 have inletwells 4152 that define cell sample inlets and outlet wells 4154 thatdefine cell sample outlets. FIG. 4B is a bottom perspective view of thesix co-joined flow-through electroporation devices of FIG. 4A alsodepicting six flow-through electroporation units 4150 arranged on asingle substrate 4156. Six inlet wells 4152 can be seen, one for eachflow-through electroporation unit 4150, and one outlet well 4154 can beseen (the outlet well of the left-most flow-through electroporation unit4150). Additionally seen in FIG. 4B are an inlet 4102, outlet 4104, flowchannel 4106 and two electrodes 4108 on either side of a constriction inflow channel 4106 in each flow-through electroporation unit 4150. Oncethe six flow-through electroporation units 4150 are fabricated, they canbe separated from one another (e.g., “snapped apart”) and used one at atime, or alternatively in embodiments where two or more flow-throughelectroporation units 4150 can be used in parallel without separation.

The flow-through electroporation devices 4150 achieve high efficiencycell electroporation with low toxicity. The flow-through electroporationdevices 4150 of the disclosure allow for particularly easy integrationwith robotic liquid handling instrumentation that is typically used inautomated systems such as air displacement pipettors. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 10 ml and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit electroporation deviceconfigurations; and integrated, automatic, multi-module cell processingand analysis.

In specific embodiments of the flow-through electroporation devices 4150of the disclosure, the toxicity level of the transformation results ingreater than 10% viable cells after electroporation, preferably greaterthan 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%,85%, 90%, or even 95% viable cells following transformation, dependingon the cell type and the nucleic acids being introduced into the cells.

The flow-through electroporation device 4150 described in relation toFIGS. 4A-4E comprises a housing with an electroporation chamber, a firstelectrode and a second electrode configured to engage with an electricpulse generator, by which electrical contacts engage with the electrodesof the electroporation device 4150. In certain embodiments, theelectroporation devices are autoclavable and/or disposable, and may bepackaged with reagents in a reagent cartridge. The electroporationdevice 4150 may be configured to electroporate cell sample volumesbetween 1 μl to 2 ml, 10 μl to 1 ml, 25 μl to 750 μl, or 50 μl to 500μl. The cells that may be electroporated with the disclosedelectroporation devices 4150 include mammalian cells (including humancells), plant cells, yeasts, other eukaryotic cells, bacteria, archaea,and other cell types.

In one exemplary embodiment, FIG. 4C depicts a top view of aflow-through electroporation device 4150 having an inlet 4102 forintroduction of cells and an exogenous reagent to be electroporated intothe cells (“cell sample”) and an outlet 4104 for the cell samplefollowing electroporation. Electrodes 4108 are introduced throughelectrode channels (not shown) in the device. FIG. 4D shows a cutawayview from the top of flow-through electroporation device 4150, with theinlet 4102, outlet 4104, and electrodes 4108 positioned with respect toa constriction in flow channel 4106. A side cutaway view of a lowerportion of flow-through electroporation device 4150 in FIG. 4Eillustrates that electrodes 4108 in this embodiment are positioned inelectrode channels 4110 and perpendicular to flow channel 4106 such thatthe cell sample flows from the inlet channel 4112 through the flowchannel 4106 to the outlet channel 4114, and in the process the cellsample flows into the electrode channels 4110 to be in contact withelectrodes 4108. In this aspect, the inlet channel 4112, outlet channel4114 and electrode channels 4110 all originate from the top planar sideof the device; however, the flow-through electroporation architecturedepicted in FIGS. 4C-4E is but one architecture useful with the reagentcartridges described herein. Additional electrode architectures aredescribed, e.g., in U.S. Ser. No. 16/147,120, filed 24 Sep. 2018; Ser.No. 16/147,865, filed 30 Sep. 2018; and Ser. No. 16/147,871, filed 30Sep. 2018.

In addition to the cell growth/concentration/transformation instrumentdescribed above with the three linked modules, additional modules may beincluded in a multi-module cell processing instrument, such as thatdepicted in FIG. 5. FIG. 5 depicts an exemplary automated multi-modulecell processing instrument 5000 comprising a TFF module 5022 asdescribed above, a flow-through electroporation device 5030 as describedabove, as well as additional exemplary modules. Illustrated is a gantry5002, providing an automated mechanical motion system (actuator) (notshown) that supplies XYZ axis motion control to, e.g., modules of theautomated multi-module cell processing instrument 5000, including, e.g.,an air displacement pipette (not shown). In some automated multi-modulecell processing instruments, the air displacement pipettor is moved by agantry and the various modules and reagent cartridges remain stationary;however, in other embodiments, the pipetting system may stay stationarywhile the various modules are moved. Also included in the automatedmulti-module cell processing instrument 5000 is wash or reagentcartridge 5004, comprising reservoirs 5006 and large and small tubes orreservoirs 5006. Wash or reagent cartridge 5004 may be configured toaccommodate large tubes, for example, wash solutions, or solutions thatare used often throughout an iterative process. In one example, wash orreagent cartridge 5004 may be configured to remain in place when two ormore reagent cartridges 5010 are sequentially used and replaced.Although reagent cartridge 5010 and wash or reagent cartridge 5004 areshown in FIG. 5 as separate cartridges, the contents of wash cartridge5004 may be incorporated into reagent cartridge 5010. Reagent cartridge5004 comprises 18 reagent vials 5012.

The exemplary automated multi-module cell processing instrument 5000 ofFIG. 5 further comprises a cell growth module 5034. In the embodimentillustrated in FIG. 5, the cell growth module 5034 comprises tworotating growth vials 5018, 5020 (described in detail with relation toFIGS. 1A-1E) as well as a TFF growth cell module 5034. There may beadditional cell concentration devices in addition to the cellconcentration capabilities of the TFF module 5034, for, e.g., cellpreparation or concentration in different cell processes. Examples ofcell concentration devices that do not utilize tangential flow includethose described in U.S. Ser. No. 16/253,564, filed 22 Jan. 2019. Alsoillustrated as part of the automated multi-module cell processinginstrument 5000 of FIG. 5 are pipet tips 5028, for use with airdisplacement pipettor (not shown), a waste repository 5026, an optionalnucleic acid assembly/desalting module 5014 comprising a reactionchamber or tube reservoir (not shown) and further comprising a magnet5016 to allow for purification of nucleic acids using, e.g., magneticsolid phase reversible immobilization (SPRI) beads (Applied BiologicalMaterials Inc., Richmond, BC).

Use of the Cell Growth Device

A general embodiment of a multi-module cell processing system is shownin FIG. 6. In some embodiments, the cell processing system 600 mayinclude a housing 660, a receptacle for introducing cells to betransformed or transfected 602, and a growth module (a cell growthdevice) 604. The cells to be transformed are transferred to the growthmodule 604 to be cultured until the cells hit a target OD. Once thecells hit the target OD, the growth module 604 may cool or freeze thecells for later processing or transfer the cells to a cell concentrationmodule 620. The cell concentration module 620 comprises the TFF deviceto generate concentrated electrocompetent cells. In one example, 20 mlof cells+growth media is concentrated to 400 μl cells in 10% glycerol.Once the electrocompetent cells have been concentrated, the cells aretransferred to a transformation module 608, such as the flow-throughelectroporation device 4150 above, to be transformed with a desirednucleic acid. In addition to the receptacle for receiving andintroducing cells into the TFF device 602, the multi-module cellprocessing system 600 includes an expression vector receptacle 606 forreceiving nucleic acids (e.g., expression or editing vectors) to betransformed or transfected into the cells. The vectors are transferredto the transformation module 608 which already contains the concentratedelectrocompetent cells grown to the specified OD, where the nucleicacids are introduced into the cells. Following transformation, the cellsare transferred into, e.g., a recovery module 610. Here, the cells areallowed to recover from the transformation process, such as anelectroporation procedure described above. In addition or alternatively,the recovery module 610 may also be used as a selection module, wherethe cells that have been transformed are selected by, e.g., antibioticsadded to the medium in the recovery module.

In some embodiments, after recovery the cells are transferred to astorage module 612 to be stored at, e.g., 4° C. or frozen. The cells canthen be retrieved from a retrieval module 614 and used for proteinexpression or further studies off-line. The automated multi-module cellprocessing system 600 is controlled by a processor 650 configured tooperate the instrument based on user input. The processor 650 maycontrol the timing, duration, temperature, and other operations(including, e.g., dispensing reagents) of the various modules of thecell processing system 600. The processor 650 may be programmed withstandard protocol parameters from which a user may select;alternatively, a user may select one or all parameters manually. Theprocessor 650 may control the wavelength at which OD is read in the cellgrowth module 604, the target OD to which the cells are grown, and thetarget time at which the cells will reach the target OD. In addition,the processor 650 may notify the user (e.g., via an application to asmart phone or other device) that the cells have reached the target ODas well as update the user as to the progress of the cells in the cellgrowth module 604 and the other modules in the multi-module system 600.Further, it should be appreciated that a stand-alone instrument mayinclude two to many cell growth devices (and cells concentration and/ortransformation devices) in parallel.

The multi-module cell processing systems 600 that comprise the cellgrowth module 604 often comprise a cell concentration module 620 (asdescribed above) and a transformation module 608 (as described above).Again, the cell concentration module 620 is configured to renderelectrocompetent and concentrate the cells that have been grown to atarget OD to an appropriate volume for cell transformation. For example,a 20 ml sample of cells in growth media is rendered electrocompetent andconcentrated to, e.g., 400 μl of cells. Concentration of the cells maybe accomplished by, e.g., the tangential flow filter (TFF) devicedescribed above, or by a centrifuge or filter, by techniques known tothose of ordinary skill in the art. Once the cells have beenconcentrated and rendered electrocompetent, the cells may be transferredto the transformation module 608.

The transformation module 608 may be configured to provide celltransformation or transfection techniques used in molecular biology. Thereagents and processing conditions for standard, routinely-usedtransformation methods may be programmed into a processor and may beselected from, e.g., a menu by a user. Alternatively, a user maymanually select reagents and processing conditions. Reagents may residewithin the transformation module 608 in bulk or may be provided with thecells as the cells are transferred from the growth module 604, forexample in tubes or vials residing in a reagent cartridge that includesa tube or vial of the cells that have been transferred to the cartridgefrom the growth module 604. Transformation of the electrocompetent cellscan take place in a flow-through electroporation device as describedabove in relation to FIGS. 4A-4E or in microfuge tubes, test tubes,cuvettes, multi-well plates, microfibers, flow systems, etc. Thetransformation module 608 preferably allows for temperature control.Control of the transformation module 608 (e.g., addition of reagents,incubation time and temperature) is typically effected by a processor650, typically a processor that controls all the modules of themulti-module cell processing instrument. It should be noted that insteadof or in addition to a transformation module 608, the multi-module cellprocessing system 600 may comprise a module configured for induction ofprotein expression and would thus comprise an induction/proteinexpression module.

The configuration of the transformation module 608 and the preprogrammedtransformation reagents and protocols depends on the transformationtechnique used. Transformation techniques include, but are not limitedto, electroporation, lipofection, optoporation, injection,microprecipitation, microinjection, liposomes, particle bombardment,sonoporation, laser-induced poration, bead transfection, calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, and the like. Additionally, hybrid techniques that exploitthe capabilities of both mechanical and chemical transfection methodscan be used, e.g., magnetofection. In another example, cationic lipidsmay be deployed in combination with gene guns or electroporators.Suitable materials and methods for transforming or transfecting targetcells can be found, e.g., in Green and Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (2014), and other laboratory manuals. Aftertransformation, the cells are allowed to recover, e.g., in a recoverymodule 610 under optimal temperature and cell media conditions, wherethe recovery module 610 also typically is under control of a processor650.

A second embodiment of a multi-module cell processing system is shown inFIG. 7. As with the embodiment shown in FIG. 6, the cell processingsystem 700 may include a housing 760, a receptacle for introducing cellsto be transformed or transfected 702, and a growth module (a cell growthdevice) 704. The cells to be transformed are transferred from the cellintroduction module 702 to the growth module 704 to be cultured untilthe cells hit a target OD. Once the cells hit the target OD, the growthmodule 704 may cool or freeze the cells for later processing or transferthe cells to a concentration module 730 where the cells are renderedelectrocompetent and concentrated to a volume optimal for celltransformation as described above in relation to the TFF device. Onceconcentrated, the cells are then transferred to the transformationmodule 708.

In addition to the receptacle for receiving cells 702, the multi-modulecell processing system 700 may include a receptacle for receivingediting oligonucleotides 716 for, e.g., nucleic acid-guided nucleaseediting, and a receptacle for receiving the editing vector backbone 718.Both the editing oligonucleotides and the editing vector backbone aretransferred to a nucleic acid assembly module 720, where the editingoligonucleotides are inserted into the editing vector backbone. Theassembled nucleic acids may be transferred into an optional purificationmodule 722 for desalting and/or other purification procedures needed toprepare the assembled nucleic acids for transformation. Once thepurification processes in the purification module 722 are complete, theassembled nucleic acids are transferred to the transformation module708, which already contains the cell culture grown to a target OD andthat has been rendered electrocompetent. In the transformation module708, the nucleic acids are introduced into the cells. Followingtransformation, the cells are transferred into a combined recovery andediting module 710. In some embodiments, the automated multi-module cellprocessing system 700 is a system that performs gene editing such as anRNA-direct nuclease editing system. For example, see U.S. Ser. No.16/024,816, filed 30 Jun. 2018; U.S. Ser. No. 16/147,865, filed 30 Sep.2018; U.S. Ser. No. 16/147,871, filed 30 Sep. 2018; U.S. Ser. No.16/269,655 filed 7 Feb. 2019; and U.S. Ser. No. 16/269,671, filed 7 Feb.2019. In the recovery and editing module 710, the cells are allowed torecover post-transformation, and the cells express the editingoligonucleotides which edit desired genes in the cells as describedbelow.

Following editing, the cells are transferred to a storage module 712,where the cells can be stored at, e.g., 4° C. until the cells areretrieved 714 for further study. The multi-module cell processing system700 is controlled by a processor 750 configured to operate theinstrument based on user input. The processor 750 may control thetiming, duration, temperature, and other operations of the variousmodules of the system 700. The processor 750 may be programmed withstandard protocol parameters from which a user may select, or a user mayspecify one or more parameters manually. The processor 750 may controlor specify the wavelength at which OD is read in the cell growth module704, the target OD to which the cells are grown, and the target time atwhich the cells will reach the target OD. In addition, the processor 750may notify the user (e.g., via an application to a smart phone or otherdevice) that the cells have reached the target OD as well as update theuser as to the progress of the cells in the cell growth module 704 andthe other modules in the multi-module cell processing system 700.

Certain embodiments of the multi-module processing system 700 such asthe system depicted in FIG. 7 include a nucleic acid assembly module 720within the system. The nucleic acid assembly module 720 is configured toaccept the nucleic acids necessary to facilitate the desired genomeediting events, and optionally the appropriate vector backbone forplasmid assembly and subsequent transformation into the cells ofinterest.

In a nuclease-directed genome editing system, a vector comprises one ormore regulatory elements operably linked to a polynucleotide sequenceencoding a nucleic acid-guided nuclease and one or more regulatoryelements are linked to a guide nucleic acid and a donor DNA comprisingthe desired edit. Alternatively, the cells may already be expressing thenuclease and the vector may comprise an editing cassette comprising theguide nucleic acid and a donor DNA. Thus, the nucleic acid assemblymodule 720 in these embodiments is configured to assemble the vectorexpressing elements contained in an editing cassette. The nucleic acidassembly module 720 may be temperature controlled depending upon thetype of nucleic acid assembly used in the instrument. For example, whenPCR is utilized in the nucleic acid assembly module 720, the moduleincludes thermocycling capability. When single temperature assemblymethods (e.g., isothermal methods) are utilized, the nucleic acid module720 is configured to have the ability to reach and hold a temperaturethat optimizes the assembly process being performed. The temperature andduration for maintaining temperatures can be effected by a preprogrammedset of parameters, or manually controlled by the user using theprocessor unit 750.

Optionally, when a nucleic acid assembly module 720 is included in amulti-module cell processing system 700, the system may also include apurification module 722 to remove unwanted components of the nucleicacid assembly mixture (e.g., salts, minerals) and optionally concentratethe assembled nucleic acids. Examples of methods for exchanging liquidfollowing nucleic acid assembly include magnetic beads (e.g., SPRI orDynal), silica beads, silica spin columns, glass beads, precipitation(e.g., using ethanol or isopropanol), alkaline lysis, osmoticpurification, extraction with butanol, membrane-based separationtechniques, filtration etc. In one aspect, the purification module 722provides filtration, e.g., ultrafiltration. For example, a range ofmicroconcentrators fitted with anisotropic, hydrophilic-generatedcellulose membranes of varying porosities may be employed. In anotherexample, purification involves contacting a liquid sample of nucleicacid and an ionic salt with an ion exchanger comprising an insolublephosphate salt, removing liquid from the nucleic acids in the sample,and eluting said nucleic acid from the ion exchanger.

In one embodiment, the automated multi-module cell processing system 700is a nuclease-directed genome editing system. Multiple nuclease-basedsystems exist for providing edits into a cell, and each can be used ineither single editing systems such as described above in relation to theautomated cell processing system 700 of FIG. 7; sequential editingsystems as could be performed in the automated system 700 of FIG. 7using the modules of the system repeatedly, e.g., using differentnuclease-directed systems sequentially to provide two or more genomeedits in a cell, and/or utilizing a single nuclease-directed systemsequentially to introduce two or more genome edits in a cell. Automatednuclease-directed processing systems can use the nucleases to cleave thecell's genome, to introduce one or more edits into a target region ofthe cell's genome, or both. Nuclease-directed genome editing mechanismsinclude zinc-finger editing mechanisms (see Urnov et al., Nature ReviewsGenetics, 11:636-64 (2010)), meganuclease editing mechanisms (see Epinatet al., Nucleic Acids Research, 31(11):2952-62 (2003); and Arnould etal., Journal of Molecular Biology, 371(1):49-65 (2007)), and RNA-guidedediting mechanisms (see Jinek et al., Science, 337:816-21 (2012); andMali et al, Science, 339:823-26 (2013)). In particular embodiments, thenuclease editing system is an inducible system that allows control ofthe timing of the editing (see Campbell, Biochem J., 473(17): 2573-2589(2016); and Dow et al., Nature Biotechnology, 33390-94 (2015)). That is,when the cell or population of cells comprising a nucleic acid-guidednuclease encoding DNA is in the presence of the inducer molecule,expression of the nuclease can occur. The ability to modulate nucleaseactivity can reduce off-target cleavage and facilitate precise genomeengineering.

EXAMPLES Example I: Growth in the Cell Growth Module

One embodiment of the cell growth device as described herein was testedagainst a conventional cell shaker shaking a 5 ml tube and an orbitalshaker shaking a 125 ml baffled flask to evaluate cell growth inbacterial and yeast cells. Additionally, growth of a bacterial cellculture and a yeast cell culture was monitored in real time using anembodiment of the cell growth device described herein.

In a first example, 20 ml EC23 cells (E. coli cells) in LB were grown ina 35 ml rotating growth vial with a 2-paddle configuration at 30° C.using the cell growth device as described herein. The rotating growthvial was spun at 600 rpm and oscillated (i.e., the rotation directionwas changed) every 1 second. In parallel, 5 ml EC23 cells in LB weregrown in a 5 ml tube at 30° C. and were shaken at 750 rpm. OD₆₀₀ wasmeasured at intervals using a NanoDrop™ spectrophotometer (Thermo FisherScientific). The results are shown in FIG. 8. The rotating growthvial/cell growth device performed better than the cell shaker in growingthe cells to OD₆₀₀ 2.6 in slightly over 4 hours. Another experiment wasperformed with the same conditions (volumes, cells, oscillation) theonly difference being a 3-paddle rotating growth vial was employed withthe cell growth device, and the results are shown in FIG. 9. Again, therotating growth vial/cell growth device performed better than the cellshaker in growing the cells to OD₆₀₀ 1.9.

Two additional experiments were performed, this time comparing therotating growth vial/cell growth device to a baffled flask and anorbital shaker. In one experiment, 20 ml EC138 cells (E. coli cells) inLB were grown in a 35 ml rotating growth vial with a 4-paddleconfiguration at 30° C. The rotating growth vial was spun at 600 rpm andoscillated (i.e., the rotation direction was changed) every 1 second. Inparallel, 20 ml EC138 cells in LB were grown in a 125 ml baffled flaskat 30° C. using an orbital shaker. OD₆₀₀ was measured at intervals usinga NanoDrop™ spectrophotometer (Thermo Fisher Scientific). The resultsare shown in FIG. 10, demonstrating that the rotating growth vial/cellgrowth device performed as well as the orbital shaker in growing thecells to OD₆₀₀ 1.0. In a second experiment 20 ml EC138 cells (E. colicells) in LB were grown in a 35 ml rotating growth vial with a 2-paddleconfiguration at 30° C. using the cell growth device as describedherein. The rotating growth vial was spun at 600 rpm and oscillated(i.e., the rotation direction was changed) every 1 second. In parallel,20 ml EC138 cells in LB were grown in a 125 ml baffled flask at 30° C.using an orbital shaker. OD₆₀₀ was measured at intervals using aNanoDrop™ spectrophotometer (Thermo Fisher Scientific). The results areshown in FIG. 11, demonstrating that the rotating growth vial/cellgrowth device performed as well—or better—as the orbital shaker ingrowing the cells to OD₆₀₀ 1.2.

In yet another experiment, the rotating growth vial/cell growth devicewas used to measure OD₆₀₀ in real time. FIG. 12 is a graph showing theresults of real time measurement of growth of an EC138 cell culture at30° C. using oscillating rotation and employing a 2-paddle rotatinggrowth vial. Note that OD₆₀₀ 2.6 was reached in 4.4 hours.

In another experiment, the rotating growth vial/cell growth device wasused to measure OD₆₀₀ in real time of yeast s288c cells in YPAD. Thecells were grown at 30° C. using oscillating rotation and employing a2-paddle rotating growth vial. FIG. 13 is a graph showing the results.Note that OD₆₀₀ 6.0 was reached in 14 hours.

Example II: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module), and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example III: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD₆₀₀ of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. An automated cell processing system comprising a cellgrowth device comprising: a housing; a motor; a thermal control device;a spectrophotometer; a processor; an electrical connection configured tobe electrically coupled to the thermal control device; and a rotatinggrowth vial wherein the rotating growth vial comprises a vial; a driveengagement mechanism configured to engage with the motor to spin thevial; and a light path through the vial for a light beam generated bythe spectrophotometer, wherein the spectrophotometer is configured tomeasure and deliver to the processor an optical density of cells in thevial; a cell concentration module; and a transformation module; whereinthe processor accepts input from a user, receives from thespectrophotometer the optical density of the cells in the vial, throughthe electrical connection directs the thermal control device to adjustthe temperature of the vial.
 2. The automated cell processing system ofclaim 1, wherein optical density is measured at a pre-programmedwavelength.
 3. The automated cell processing system of claim 1, whereinoptical density is measured at a wavelength selected by a user.
 4. Theautomated cell processing system of claim 1, wherein the optical densityis measured continuously.
 5. The automated cell processing system ofclaim 1, wherein the optical density is measured at intervals specifiedby the user or programmed into the processor.
 6. The automated cellprocessing system of claim 1, wherein the processor notifies a user whena desired optical density is reached.
 7. The automated cell processingsystem of claim 1, wherein the vial volume is 2-100 ml.
 8. The automatedcell processing system of claim 1, wherein the cells are grown to atarget optical density value at a target time.
 9. The automated cellprocessing system of claim 1, wherein the vial is fabricated frompolycarbonate or polypropylene.
 10. The automated cell processing systemof claim 1, wherein the transformation module comprises a flow-throughelectroporation device.
 11. An automated cell processing systemcomprising: a cell growth device comprising a housing; a motor; athermal control device; a spectrophotometer; a processor; an electricalconnection configured to be electrically coupled to the thermal controldevice; and a disposable rotating cell growth vial wherein thedisposable rotating growth vial comprises a vial; a drive engagementmechanism connected to the motor and configured to spin the vial; and afirst light path through the vial to measure an optical density of cellsin the vial via the spectrophotometer; a transformation module; and anediting module; wherein the processor accepts input from a user,receives from the spectrophotometer the optical density of the cells,and through the electrical connection directs the thermal device toadjust the temperature of the vial to grow the cells.
 12. The automatedcell processing system of claim 11, wherein the optical density ismeasured at a pre-programmed wavelength.
 13. The automated cellprocessing system of claim 11, wherein the optical density is measuredat a wavelength selected by a user.
 14. The automated cell processingsystem of claim 11, wherein the optical density is measuredcontinuously.
 15. The automated cell processing system of claim 11,wherein the optical density is measured at intervals specified by theuser or programmed into the processor.
 16. The automated cell processingsystem of claim 11, wherein the processor notifies a user when a desiredoptical density is reached.
 17. The automated cell processing system ofclaim 11, wherein the cells are grown to a target optical density valueat a target time.
 18. The automated cell processing system of claim 11,wherein the transformation module comprises a flow-throughelectroporation device.
 19. The automated cell processing system ofclaim 11, further comprising a liquid handling system controlled by theprocessor.
 20. The automated cell processing system of claim 11, whereinthe vial further comprises a second light path.